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Title: Developing integrated methods for probing flexible, hidden and metastable protein states : from side chains to global dynamics
Author: Pritchard, Ruth Bronwen
ISNI:       0000 0004 7660 1216
Awarding Body: UCL(University College London)
Current Institution: University College London (University of London)
Date of Award: 2018
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Understanding the structure and dynamics of biological systems at an atomic level is key to characterising their function and pathology. Many existing methods focus largely on obtaining snap shots of static protein structures and have limited scope to capture the wide variety of motions that occur. Dynamics, particularly in the chemically-diverse side chains, often play fundamental roles in biological events including folding, binding and catalysis. There is an increasingly evident need for methods elucidating the behaviour of side chains and exible systems in general. This thesis presents both new NMR spectroscopy methods probing side-chain conformational dynamics, and a comparison of the performance of molecular dynamics force elds simulating a exible protein system. First, both protonand carbon-detected pulse sequences to record long-range J -coupling constants in lysine and arginine residues are reported and used to probe χ-angle sampling. Importantly, these results indicate the possibility of developing methods where chemical shifts alone can be used to obtain similar information. Secondly, a new class of carbon-detected experiments is presented for the measurement of side-chain carbon-carbon correlations in large proteins (∼82kDa). It is also shown that the carbon-detected pulse sequences to measure J -coupling constants can be used to probe side-chain sampling in constructs with high molecular weight. These results highlight the signi cant bene ts of carbon detection for large systems and indicate strong potential for the development of a suite of experiments recording a wide range of parameters. Finally, an extensive NMR-derived data set was used to assess the performance of eight state-of-the-art force elds simulating a exible protein construct with very mixed structural composition. Generally, these force elds characterised the well-folded regions correctly but struggled to represent the exible, partially-structured regions. Overall, the presented work builds into the larger body of integrated approaches for characterising molecular systems in the context of their dynamics as well as structure and function.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available